Table 1.
Patient details including clinical status and clinical atopy.
Figure 1.
Measurement of Transepithelial Electrical Resistance (TEER).
A - TEER of unstimulated PNECs and PBECs cultured for 28 d. Values are expressed as mean ± SD. No significant difference was observed in the TEER values between the groups. B - TEER of PNECs and PBECs cultured with 20 ng/ml IL-13 for 28 d. Values are expressed as mean ± SD. On days 7 and 14 there was a significant difference in TEER values between PNECs and PBECs (p<0.02 for each time point) however by the end of the culture period the TEER values were similar between groups.
Table 2.
Total cell count on day 28 of ALI culture.
Figure 2.
Quantification of Goblet Cell Number.
Number of goblet cells from PNECs and PBECs treated with 20/ml IL-13 expressed as the percentage differential goblet cell count corrected for cell number on d 28 of ALI culture. Comparisons of average values between groups were performed using paired t-tests. There was a significant difference seen between unstimulated and IL-13 stimulated PNECs (p = 0.0001) and in IL-13 stimulated PBECs when compared to unstimulated PBECs (p = 0.0036). Additionally, there was a significant difference between PNEC unstimulated and PBEC unstimulated (p = 0.033) and when cells where stimulated with IL-13, the percentage of goblet cells was significantly higher in stimulated PBECs compared to stimulated PNECs (p = 0.009).
Figure 3.
Gene expression of SPDEF & MUC5AC mRNA and ELISA for MUC5AC mucin secretion.
A - Gene expression of SPDEF mRNA using comparative quantitation real time PCR in IL-13 stimulated PNECs and PBECs expressed as fold change compared to unstimulated cells. In both cell types, stimulation with IL-13 caused a significant increase in SPDEF mRNA levels (PNECs: p = 0.013; PBECs: p = 0.02). B - Gene expression of MUC5AC mRNA using comparative quantitation real time PCR in IL-13 stimulated PNECs and PBECs expressed as fold change compared to unstimulated cells. There was no significant increase in MUC5AC mRNA levels in PNECs, however, stimulation with IL-13 caused a significant increase in MUC5AC mRNA levels in PBECs (p = 0.04). C - Relative optical density (OD λ = 450 nm) of MUC5AC secreted apically using ELISA corrected for MUC5AC positive control. There was no significant difference between unstimulated and IL-13 stimulated PNECs or PBECs.
Figure 4.
Quantification of Ciliated Cells.
Number of ciliated cells from PNECs and PBECs treated with 20/ml IL-13 expressed as the percentage differential ciliated cell count corrected for cell number on d 28 of ALI culture. Comparisons of average values between groups were performed using paired t-tests. In unstimulated conditions, PBEC cultures contained a significantly higher percentage of ciliated cells than PNECs (p = 0.0001). However, when stimulated with IL-13, the percentage of ciliated cells significantly decreased in PBECs (p = 0.005), but IL-13 had no effect on ciliated cell numbers in PNECs. Additionally, IL-13 stimulated PBECs had significantly higher numbers of ciliated cells compared with IL-13 stimulated PNECs (p = 0.002).
Figure 5.
Cytokine Analysis of Apical Washings and Basolateral Supernatants using Bioplex.
A – Apical secretion of cytokines in PBEC and PNEC cultures. Results were corrected for cell numbers and a fold change ratio was calculated (IL-13 stimulated/unstimulated). In order to graphically represent all detectable cytokines the results were plotted on a logarithmic axis. There were no significant differences in any of the detected cytokines secreted between PNECs and PBECs. B - Basolateral secretion of cytokines in PBEC and PNEC cultures. Results were corrected for cell number and a fold change ratio was then calculated (IL-13 stimulated/unstimulated). In order to graphically represent all detectable cytokines the results were plotted on a logarithmic axis. There were no significant differences in any of the cytokines secreted between PNECs and PBECs.